By means of a monoclonal antibody (mab) against the rat liver nuclear L-T3 receptor (NT3R) and a polyclonal anti-GFAp serum, it has been possible to demonstrate nuclear thyroid hormone receptors in astrocyte cultures. On day 3, 47% of GFAp+ cell nuclei were labeled by 2B3 mab. Between day 3 and day 15, the number of GFA+ cell nuclei stained by 2B3 mab increased from 47 to 75%. Thyroid hormone nuclear receptors were present in fibrous and protoplasmic astrocytes. However, they developed asynchronously in both types of astrocytes. Indeed, 60% of fibrous astrocytes were stained by 2B3 mab on day 3 and this percentage reached 77% after 8 days in vitro. In contrast, only 30% of protoplasmic astrocytes were immunoreactive for 2B3 mab on day 3 and this percentage increased slowly reaching 47% on day 8 and around 75-80% on day 15. By immunoblotting, the monoclonal antibody recognized two bands of proteins with a molecular weight of 57 and 45 kDa respectively. These proteins have the same electrophoretic mobility as [125I]bromoacetyl-LT3 rat liver nuclear L-T3 receptor. This paper presents the first immunocytochemical localization of nuclear L-T3 receptors in astrocyte cultures. Furthermore, we show that thyroid hormone receptors develop more rapidly in fibrous than in protoplasmic astrocytes.